The present relates to amperometric biosensors capable of rapidly quantifying the concentration of an analyte in a sample solution with high accuracy in a simplified manner, and to methods of producing same. In particular, the invention relates to amperometric biosensors having an immobilized, redox enzyme as a enzymatic sensing element coupled to a metal electrode.
A biosensor is an analytical device incorporating biological and chemical sensing elements, either intimately connected to or integrated with a suitable transducer, which enables the conversion of concentrations of specific chemicals into electronic signals. A majority of biosensors produced thus far have incorporated an enzyme as a biological recognition component.
A critical element in the design of a successful biosensor is the immobilization procedure for biological components. Generally, four main approaches to enzyme immobilization have been utilized. These include: (1) physical adsorption at a solid surface, (2) entrapment in polymeric gels or microcapsules, (3) cross-linking by means of bifunctional reagents, and (4) covalent binding to a reactive insoluble support. Although these methods are generally used in the construction of biosensing devices, specific details in the immobilization and assembly protocols also may be crucial to the development of reliable, as well as sensitive biosensors.
This main challenge in biosensor technology is to find an effective means to couple the biological component to the transducer. This coupling is particularly important to the development of amperometric biosensors, since conventional metal electrodes are generally very poor voltammetric electrodes for the direct oxidation or reduction of biological components. The approaches used to modify these electrodes for use as biosensors can be divided into two groups: (1) modification of the electrode surface by deposition of a monolayer, which is based upon either the adsorption of a species at the electrode surface or a covalent attachment of redox mediators to the electrode, and (2) modification by a multilayer, which is most frequently achieved by the use of polymeric modifications of the electrode. Here too, specific details of the modification procedures may be crucial to the development of useful biosensing devices.
Mediators are also frequently used in the final biosensing device. Due to the inaccessible nature of the redox centers of oxido-reductase enzymes, mediators or electron shuttles are added to biosensors either by physically admixing the mediator with the enzyme or by chemically binding the mediator to the enzyme to enhance electron transfer from a reactant or desired analyte through the enzyme to the electrode. For example, mediated glucose sensors involving electron acceptors, such as ferricyanide, quinones, and various organic dyes have been utilized.
The goal of a particular biosensing device is to accurately measure a specific biological substrate or analyte within a sample solution. For example, a reliable fructose sensor could be of use for the quantitation of the sugar in food products such as fruit juice, high fructose corn syrup and wine, as well as in clinical samples including blood serum and seminal plasma. Although an enzymatic spectrophotometric assay is available for fructose determination, the assay is time intensive, tedious and costly.
Several groups recently have described the immobilization of Gluconobacter sp. fructose dehydrogenase, a 140 kDa, membrane-bound, pyrroloquinoline quinone-containing oxidoreductase, or various electrodes to give potential fructose biosensors. This fructose sensing enzyme has also been immobilized on glassy carbon, gold and platinum by entrapping it in conductive polypyrrole matrices on platinum and coupling it with the organic conducting salt TTF-TCNQ in a polypyrrole matrix on glassy carbon. Alternatively, the enzyme has been secured on a carbon paste electrode with and without mediators. This fructose sensing enzyme also has been immobilized within a cell-membrane mimetic environment on gold in the presence of a mediator. While these approaches delivered some promising results, readily oxidizable inteferents such as ascorbic acid in citrus juice were found to overwhelm the fructose signal. When this problem is partially avoided by using lower redox potentials, relatively poor sensitivity and low current response is the result.
Other biological analytes of interest are creatinine, creatine and sarcosine. Creatinine is the final product of creatine metabolism in mammals. During kidney dysfunction or muscle disorder, the creatine concentration in serum/plasma may rise to levels several fold the norm. The measurement of the creatinine levels in serum and the determination of the renal clearance are widely used for laboratory diagnosis of renal and muscular function. Most creatine measurements, however, rely on spectrophotometric procedures based on the Jaffe reaction. These assays are analytically limited in that the Jaffe reaction is not specific for creatinine. Given this, many other substrates interfere with the assay leading to inaccurate determinations of creatine concentration in the sample.
In the case of creatinine biosensors, these sensors were first described by Meyerhoff and Rechnitz in 1976. Since that time, many enzymatic creatine sensor systems have been developed. More recent work on creatinine biosensors has utilized a sequence of three enzymes with sarcosine as the final enzyme which requires oxygen to reoxidize the enzyme. In this example, the presence of creatinine was detected by amperometric measurement of concentration changes of either oxygen consumed, or hydrogen peroxide, formed in the final reaction. These methods, however, are rather complex and inefficient because there is a stringent requirement for precise control of oxygen concentration in the system. It is inconvenient, for example, to ensure that oxygen concentration in a series of blood samples is maintained at a constant. Additionally, the electrodes for hydrogen peroxide detection require high overpotentials which may cause blood metabolites, such as ascorbic acid or uric acid, to be oxidized at the electrodes, thus leading to inaccurate measurements.
In summary, although the prior art teaches the use of amperometric biosensing systems as tools to accurately measure biological analytes of interest, many problems arise in the application of these biosensors, such as the relative sensitivity, selectivity and stability of the sensing device.
In particular, some systems are prone to inaccuracies due to the presence of interfering agents present in the test samples. One example, is the presence of ascorbate in fruit juice. The ascorbate acts an interferent and leads to biosensing devices that are inaccurate in terms of the concentration of the measured analyte. Another problem arises from uncontrolled oxygen concentration in some biosensing systems designed to measure creatinine. Thus, there exists a need for biosensors which are highly selective sensitive, and not prone to interference by other chemicals present in the sample. Finally, it is desired that these biosensors also display increased stability, thus allowing for repeated use of the sensing electrode.